Special Adenovirus Production Services for Toxic Genes

Amplification and Purification of Adenovirus (1 ml, 1x10e12 vp/ml)

Welgen provides a customer 3 ml of 1×1012 viral particles/ml in our standard amplification/purification services (CAT#3000). If a customer needs less amount of adenoviral stock, Welgen also can do it for you. Existing adenovirus vectors supplied by our customers or newly constructed vectors will be amplified to 1 x 1012 viral particles. The adenovirus will then be purified by centrifugation using two sequential cesium chloride gradients. The final product (1 ml, 1×1012 viral particles/ml) will be dialyzed, tittered spectrophotometrically, and tested for sterility, and then shipped to customer. The service time is approximately 2 weeks.

Amplification and Purification of Adenovirus (3 ml, 1x10e12 vp/ml)

Existing adenovirus vectors supplied by our customers or newly constructed vectors will be amplified to obtain more than 3 x 1012 viral particles, which requires approximately 2 weeks. The adenovirus will then be purified by centrifugation using two sequential cesium chloride gradients. The final product (3 ml, 1×1012 viral particles/ml) will be dialyzed, tittered spectrophotometrically, and tested for sterility, and then shipped to customer.

shRNA Selection Service

Three short hairpin RNA (shRNA) sequences will be selected and cloned into a pQuietU6 plasmid. The desired constructs will be sequenced to verify the correct shRNA. The 3 shRNA plasmids will then be co-transfected into 293 cells with an cDNA expression plasmid. The most effective shRNA can be determined by a Western blot. Welgen guarantees that the selected vector will provide 70% silencing efficiency in a co-transfection experiment.

Construct an Adenovirus to Silence Gene Expression

The customer will provide Welgen an effective shRNA sequence. Welgen will be synthesized and inserted it into pQuietU6 vector and convert it into an adenovirus. A crude cell lysate (106 vp/ml) can be shipped to customer within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks.

Construct a pQuiet Vector to Silence Gene Expression

The customer will provide us a validated shRNA (short hairpin RNAi) sequence. Welegn will synthesize a double-stranded oligonucleotide that encodes the validated shRNA sequence and inserted into a pQuiet vector. The vector will be amplified and sequenced to ensure that the correct shRNA sequence will be expressed. You will receive your plasmid within 2 weeks. If you would like more than one sequence to be inserted into pQuiet 2, 3, or 4 for multiple gene silencing, the additional cost will be charged per sequence. All you need to do is to provide the validated, effective 19 bp sequences from the target genes.

Retrovirus Titering Assay

In this service, the titer of retrovirus (provided by customer or constructed by Welgen) is determined using 293 cells.
The assay usually takes 2 weeks to finish.

Customer Retroviral RNAi Services

RNAi design, selection, validation
Guarantee 70% silencing effect in a cotransfection experiment
Retrovirus construction and production
Delivery of 100 ul of concentrated viral stocks (approximately 1 x 108-9 TU/ml)

Customer Retroviral Construction and Production

Customer provides the retroviral vectors and cDNA
Welgen will clone the cDNA into the retroviral vectors
Welgen produces high titer of lentivirus (100 ul concentrated viral stocks, approximately 1 x 108-9 TU/ml)

Large-scale Retrovirus Production

Customer submits 3 mg DNA prepared by Qiagen kit
Welgen delivers 1 ml of concentrated viral stocks (approximately 1 x 108-9 TU/ml)

Standard Retrovirus Production

Customer submits 100 ug DNA prepared by Qiagen Kit
Welgen delivers 100 ul of concentrated viral stocks (approximately 1 x 108-9TU/ml)

Mini-scale Retrovirus Production

Customer submits 10 ug DNA prepared by Qiagen Kit
Welgen delivers 2-4 ml of virus
Concentrated viral stock is not available for this service

MOI Assay

The multiplicity of infection or MOI is the ratio defined by the number of infectious virus particles divided by the number of target cells. In this service, MOI will be assayed using cells (provided by customer or by Welgen). It usually takes two to three weeks.

Isolate Adenoviral DNA Sample

More than 5 ug high quality adenoviral DNA will be purified from approximately 10¹¹viral particles.

TCID50

HEK293 cells growing on 96 well plates will be infected with serial dilutions of an adenoviral vector stock and the resulting infection pattern will be evaluated to determine the TCID50 (Tissue Culture Infectious Dose 50) using the Karber formula.

RCA Assay in A549 Cells

The standard method to determine the presence of RCA (Replication-Competent Adenovirus) is to inoculate A549 cells with a sample of the viral stock. Since A549 cells are very readily infected by adenovirus but do not express the E1 gene, no plaque will form unless viral particles that have acquired the E1 gene are present. The sensitivity of this method is limited by the cytotoxic effects of the replication-deficient virus on the A549 cells, which limits the number of particles that can be added to the cultured cells. Approximately 1 RCA per 10⁶ viral particles (determined spectrophotometrically) can be detected by this method.

PCR Assay for RCA

The DNA purified from viral particles is used as a template for a PCR procedure in which E1 gene specific primers are used to detect the presence of the E1 gene. The sensitivity of this method is limited by the amount of template DNA that can be added. Greater sensitivity (10-fold) can be achieved by doing 10 replicate PCR reactions. The PCR procedure to detect the presence of RCA is faster and more sensitive than the assay that employs A549 cells and is recommended before amplification of a viral stock.

Plaque Assay to Titer Adenovirus

The abundance of adenoviral particles can be determined in a variety of ways. Two of the most useful are the spectrophotometric method, which is fast and highly reproducible, and the plaque assay, which estimates the number of infectious particles that are present. The plaque assay is performed by treating host cells in which the virus can replicate with serial dilutions of the virus preparation for several days. During the incubation, infected cells grow new viral particles that infect neighboring cells, which die and release more adenovirus until a plaque becomes visible. The plaque assay is slower and less reproducible than the spectrophotometric assay, but it provides a result that may be a more realistic estimate of the number of viral particles needed to evoke a biological response. A normal ratio between the spectrophotometric and plaque assay results indicates that a purified viral product is not contaminated with UV absorbent material.

Plaque Purification

Newly generated vectors that have been constructed using our adenoviral system do not require plaque purification, but when previously amplified vectors are re-amplified, rare crossover events can result in the appearance of RCA (Replication-Competent Adenovirus), which must then be eliminated by plaque purification. Recombination is expected to occur only after adenovirus has been amplified extensively and generally results in replacement of the desired transgene with the E1 gene that was deleted to make the virus replication-deficient. Amplification of viral stocks contaminated by RCA results in overgrowth by the RCA. It is always prudent to perform an RCA assay (#S3130 or S3140) before re-amplifying a viral stock.

Amplification and Purification of Adenovirus (1 ml)

Existing adenovirus vectors supplied by our customers or newly constructed vectors will be amplified to obtain more than 3 x 10¹² viral particles, which requires approximately 2 weeks. The adenovirus will then be purified by centrifugation using two sequential cesium chloride gradients. The final product (3 ml, 1×10¹² viral particles/ml) will be dialyzed, tittered spectrophotometrically, and tested for sterility, and then shipped to customer.

Amplification and Purification of Adenovirus (3 ml)

Welgen provides a customer 3 ml of 1×10¹² viral particles/ml in our standard amplification/purification services (CAT#3000). If a customer needs less amount of adenoviral stock, Welgen also can do it for you. Existing adenovirus vectors supplied by our customers or newly constructed vectors will be amplified to 1 x 10¹² viral particles. The adenovirus will then be purified by centrifugation using two sequential cesium chloride gradients. The final product (1 ml, 1×10¹² viral particles/ml) will be dialyzed, tittered spectrophotometrically, and tested for sterility, and then shipped to customer. The service time is approximately 2 weeks.

Generation of Adenovirus (adenoviral construct provided by customer)

By using this service, the customer provides the adenovirus construct and Welgen will generate adenovirus in 293 cells. Welgen will deliver a crude cell lysate containing the viral construct (approximately 10⁶ viral particles/ml) within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks.

shRNA Selection and Validation Service

The customer will provide Welgen an effective shRNA sequence. Welgen will be synthesized and inserted it into pQuietU6 vector and convert it into an adenovirus. A crude cell lysate (10⁶ vp/ml) can be shipped to customer within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks.

Construct an Adenovirus to Silence Gene Expression

The customer will provide Welgen an effective shRNA sequence. Welgen will be synthesized and inserted it into pQuietU6 vector and convert it into an adenovirus. A crude cell lysate (10⁶ vp/ml) can be shipped to customer within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks.

Construct a pQuiet Vector to Silence Gene Expression

The customer will provide us a validated shRNA (short hairpin RNAi) sequence. Welgen will synthesize a double-stranded oligonucleotide that encodes the validated shRNA sequence and inserted into a pQuiet vector. The vector will be amplified and sequenced to ensure that the correct shRNA sequence will be expressed. You will receive your plasmid within 2 weeks. If you would like more than one sequence to be inserted into pQuiet 2, 3, or 4 for multiple gene silencing, the additional cost will be charged per sequence. All you need to do is to provide the validated, effective 19 bp sequences from the target genes.

Lentivirus Titering Assay

In this service, the titer of lentivirus (provided by customer or constructed by Welgen) is determined using 293 cells.
The assay usually takes 2 weeks to finish.

Customer Lentiviral RNAi Services

RNAi design, selection, validation
Guarantee 70% silencing effect in a cotransfection experiment
Lentivirus construction and production
Delivery of 100 ul of concentrated viral stocks (approximately 1 x 108-9 TU/ml)

Customer Lentiviral Construction and Production

Customer provides the lentviral vectors and cDNA.
Welgen will clone the cDNA into the lentiviral vectors
Welgen produces high titer of lentviirus (100 ul concentrated viral stocks, approximately 1 x 108-9 TU/ml)

Large-scale Lentivirus Production

Customer submits 3 mg DNA prepared by Qiagen kit
Welgen delivers 1 ml of concentrated viral stocks (approximately 1 x 108-9 TU/ml)

Standard Lentivirus Production

Customer submits 100 ug DNA prepared by Qiagen Kit
Welgen delivers 100 ul of concentrated viral stocks (approximately 1 x 108-9TU/ml)

Mini-scale Lentivirus Production

Customer submits 10 ug DNA prepared by Qiagen Kit
Welgen delivers 2-4 ml of virus
Concentrated viral stock is not available for this service

Standard AAV Production

For this service:

Multiple serotypes ( AAV2, AAV5, AAV8, AAV9, AAV-DJ).
Medium scale transfection of AAV293 cells into 40×150 mm plates.
Harvest rAAV followed by purification via a iodixanol or CsCl density gradient centrifugations.
Titration (measuring genome copies).

Required Materials:

AAV cis plasmid carrying your gene of interest (GOI), >150 ug.

Turnaround Time:

2-3 weeks.

Deliverables:

2 ml purified viral stocks (approximately 1 x 1012-13) GC/ml).

Final viral yield may depend on the nature of transgene.

Customer AAV Construction and Production

For this service:

Multiple serotypes ( AAV2, AAV5, AAV8, AAV9).
Wide choice of AAV cis vectors (promoterless, CMV, PGK, UBC or U6 promoters with or without reporter genes like GFP, mCherry, strawberry, Luc, LacZ, etc).
Medium scale transfection of AAV293 cells into 20 x150 mm plates.
Harvest rAAV followed by purification via a serial of CsCl centrifugations.
Titration (measuring genome copies).

Required Materials:

Validated shRNA sequence or a NCBI accession # or plasmid DNA carrying your gene of interest (GOI).

Turnaround Time:

2-3 weeks.

Deliverables:

2 ml purified viral stocks (approximately 1 x 1012-13 GC/ml).

Construct an Adenovirus Vector to Express a Gene (target cDNA not provided by customer)

If a customer does not provide the cDNA, Welgen will clone the cDNA or purchase the cDNA for the customer. The cDNA will be inserted into our adenovirus system and generate adenovirus. A crude cell lysate containing the viral construct (approximately 10⁶ viral particles/ml) can be delivered to the customer within 3 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat# S3000) will require another 2 weeks.

Construct an Adenovirus Vector to Express a Gene (target cDNA provided by customer)

Welgen will use insert your sequence into the adenoviral genome and return a crude cell lysate containing the viral construct (approximately 10⁹ viral particles/ml) within 2 weeks. The crude cell lysate is suitable for testing to ensure that the virus performs as expected. Additional amplification and purification of the virus (Cat#S3000) will require another 2 weeks. Typically we will need about 10 ug of a plasmid containing the gene to be expressed.

Service ID	Service	Estimated Price*
ST1100	Construct an Adenovirus (customer provides cDNA)
ST3000	Amplification and purification of adenovirus (3 ml, 1x10e12 vp/ml)
ST3010	Amplification and purification of adenovirus (1 ml)